Amicon ultra 100k filter

Expression and purification of recombinant EhV-ATPase were performed as previously described^{41 (link)}. In brief, E. hirae V-ATPase was expressed recombinantly in Escherichia coli and purified by affinity purification with a Ni⁺-NTA (nitrilotriacetic acid) column (Ni⁺-NTA Superflow; Qiagen, Hilden, Germany). The column was preconditioned with buffer consisting of 50 mM potassium phosphate, 100 mM KCl, 5 mM MgCl₂, 20 mM imidazole, 10% glycerol, 0.05% n-dodecyl-β-D-maltoside (βDDM) at pH 7.5. The column was washed, and protein was eluted with buffer containing 50 mM potassium phosphate, 100 mM KCl, 5 mM MgCl₂, 300 mM imidazole, 10% glycerol, 0.05% βDDM at pH 7.5. The purified protein was concentrated using an Amicon Ultra 100 K filter (Merck Millipore, Billerica, Massachusetts, USA) before further purification using Superdex 200 gel filtration (GE Healthcare, Little Chalfont, UK) preconditioned with 50 mM Tris-HCl, 5 mM MgCl₂, 10% glycerol, 0.05% βDDM at pH 7.5. The rotor-rotating activity of the EhV-ATPase in presence of ATP, Na⁺, Mg²⁺ was confirmed by inhibition of ATP hydrolysis of the V₁ domain by DCCD, an inhibitor of c-ring rotation in the V_o domain^{33 (link)}.

293T cells stably producing SVPs were incubated in Opti-MEM (Invitrogen). Supernatant was filtered through a 0.45-μm filter to remove cell debris, then concentrated by ultrafiltration using an Amicon Ultra 100K filter (Merck Millipore). Concentrated supernatant was fractionated by chromatography using Superdex 200 (GE). Fractions containing SVPs were pooled for use in immunization.

Isolation of rituximab was performed with M-280 tosylactivated magnetic beads (Life Technologies, California, USA) according to the protocol provided by the supplier. In brief, 200 μg of anti-rituximab antibody (MB2A4) (Gene Tex, California, USA) were covalently attached to 10 mg of magnetic beads. Then, the free tosylate groups of the beads were blocked by incubation with PBST containing 1% BSA for 1 h. Rat plasma samples were concentrated using an Amicon^® Ultra 100K filter (Merck Millipore, Massachusetts, USA). The concentrated plasma was incubated with the beads for 3 h at room temperature. After washing, rituximab was eluted with 0.1 M glycine-HCl (pH 2.7).
For the CDC and ADCC assays, plasma samples were purified to isolate rituximab by an Ex-Pure Spin ProA (Kyoto Monotech, Kyoto, Japan) centrifuge following the protocol provided by the supplier.